Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional ...profiles of 131,224 single cells from peripheral and intra-tumoral immune populations from patients with HPV– and HPV+ HNSCC and healthy donors. Immune cells within tumors of HPV– and HPV+ HNSCC displayed a spectrum of transcriptional signatures, with helper CD4+ T cells and B cells being relatively divergent and CD8+ T cells and CD4+ regulatory T cells being relatively similar. Transcriptional results were contextualized through multispectral immunofluorescence analyses and evaluating putative cell-cell communication based on spatial proximity. These analyses defined a gene expression signature associated with CD4+ T follicular helper cells that is associated with longer progression-free survival in HNSCC patients. The datasets and analytical approaches herein provide a resource for the further study of the impact of immune cells on viral- and carcinogen-induced cancers.
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•Single-cell RNA-seq revealed distinct immune profiles in HPV– and HPV+ HNSCC•B cells, myeloid cells, and CD4+ Tconv cells were divergent by high-dimensional analysis•Multispectral imaging uncovered immune structures (TLSs) associated with HPV+ disease•T follicular helper signature was associated with favorable survival in TCGA patients
Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or human papillomavirus (HPV). Cillo et al. determine single-cell transcriptional profiles of peripheral and intra-tumoral immune populations from patients with HPV– and HPV+ HNSCC and healthy donors. These datasets, accompanied by spatial organization information, provide a resource for further study of immunity to viral- and carcinogen-induced cancers.
Current immunotherapy paradigms aim to reinvigorate CD8
T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck ...squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV
), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV
HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV
HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies.
Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of ...more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)
and 12 HPV
HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV
TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.
Immunotherapy can reinvigorate dormant responses to cancer, but response rates remain low. Oncolytic viruses, which replicate in cancer cells, induce tumor lysis and immune priming, but their immune ...consequences are unclear. We profiled the infiltrate of aggressive melanomas induced by oncolytic Vaccinia virus using RNA sequencing and found substantial remodeling of the tumor microenvironment, dominated by effector T cell influx. However, responses to oncolytic viruses were incomplete due to metabolic insufficiencies induced by the tumor microenvironment. We identified the adipokine leptin as a potent metabolic reprogramming agent that supported antitumor responses. Leptin metabolically reprogrammed T cells in vitro, and melanoma cells expressing leptin were immunologically controlled in mice. Engineering oncolytic viruses to express leptin in tumor cells induced complete responses in tumor-bearing mice and supported memory development in the tumor infiltrate. Thus, leptin can provide metabolic support to tumor immunity, and oncolytic viruses represent a platform to deliver metabolic therapy.
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•Single-cell sequencing reveals immune remodeling in Vaccinia-virus-treated tumors•The adipokine leptin enhances T cell metabolic function in vitro and in vivo•Vaccinia engineered to express leptin metabolically enhances antitumor responses•Leptin-engineered Vaccinia promotes a memory response upon tumor rechallenge
Metabolic insufficiency is a major barrier for antitumor immunity. Rivadeneira et al. demonstrate that engineering an oncolytic virus to express a metabolic modulator, in this case the adipokine leptin, improves T cell metabolic function in the tumor microenvironment, allowing a superior antitumor response compared to a control oncolytic.
Systemic sclerosis (SSc) is a devastating autoimmune disease characterized by excessive production and accumulation of extracellular matrix, leading to fibrosis of skin and other internal organs. ...However, the main cellular participants in SSc skin fibrosis remain incompletely understood. Here using differentiation trajectories at a single cell level, we demonstrate a dual source of extracellular matrix deposition in SSc skin from both myofibroblasts and endothelial-to-mesenchymal-transitioning cells (EndoMT). We further define a central role of Hippo pathway effectors in differentiation and homeostasis of myofibroblast and EndoMT, respectively, and show that myofibroblasts and EndoMTs function as central communication hubs that drive key pro-fibrotic signaling pathways in SSc. Together, our data help characterize myofibroblast differentiation and EndoMT phenotypes in SSc skin, and hint that modulation of the Hippo pathway may contribute in reversing the pro-fibrotic phenotypes in myofibroblasts and EndoMTs.
SSc is a disease characterized by inflammation and fibrosis. Heme Oxygenase-1 (HO-1) is a haem-degrading enzyme that mediates resolution of inflammation and is induced upon mediators abundantly ...present in SSc. We aimed to assess whether HO-1 expression/function is disturbed in SSc patients and could therefore be contributing to the ongoing inflammation.
In total, 92 SSc patients and 48 healthy controls were included. By measuring total bilirubin in plasma in vivo, HO-activity was assessed. HO-1 expression levels were determined with western blot in monocytes before and after induction of HO-1 with cobalt protoporphyrin (CoPP) with or without CXCL4. Monocyte-derived dendritic cells (DCs) were stimulated with several Toll-like receptor (TLR) ligands with or without pre-stimulation with CoPP for 24 h. Cytokine levels were measured in the supernatants using the Luminex Bead Array.
SSc patients have lower plasma levels of bilirubin, suggestive of an aberrant HO-1 function. We demonstrated low HO-1 expression in immune cells from SSc patients, whereas induction with CoPP was able to restore HO-1 levels in DCs from SSc patients, almost normalizing the increased TLR response observed in SSc. Co-exposure to CXCL4 completely abrogated CoPP-induced HO-1 expression, suggesting that the high CXCL4 levels present in SSc patients block the normal induction of HO-1 and its function.
We demonstrate that HO activity in SSc patients is decreased and show its functional consequences. Since CXCL4 blocks the induction of HO-1 expression, neutralization of CXCL4 in SSc patients could have clinical benefits by diminishing overactivation of immune cells and other anti-inflammatory effects of HO-1.
Abstract
B cells can regulate immune responses by presenting antigen, producing antigen-specific antibodies and immunomodulatory cytokines. Their role in the anti-tumor immune response is poorly ...understood. However, in many cancers including head and neck squamous cell carcinoma (HNSCC), intratumoral B cells correlate with better survival. HNSCC has two distinct etiologies (HPV−) and (HPV+) where patients who are HPV+ have increased B cell infiltration and respond better to therapy. We hypothesized that (1) intratumoral B cell phenotype is different between HPV+ and HPV− HNSCC (2) location within the tumor microenvironment (TME) is distinct and (3) antibodies produced by intratumoral B cell in HPV+ HNSCC are specific for viral antigens.
Using single-cell RNA sequencing and spectral flow cytometry, we observed that B cell signatures in HPV− HNSCC patients were predominantly memory B cells and plasma cells, while the signatures in HPV+ HNSCC were naïve and germinal center (GC) B cells. Further, we quantified B cells in tertiary lymphoid structures (TLS) using multispectral immunofluorescence, and the presence of GC-rich TLS were increased in HPV+ patients. In fact, GC-rich TLS within the tumor of HPV+ patients correlated with increased overall survival. Overall, high enrichment for GC B cells were positively associated with longer progression-free survival. Antibodies produced by intratumoral B cells from HPV+ patients were positive for HPV viral antigens. Ultimately, characterization of B cell phenotype and function in HNSCC is important for devising new therapeutic options for cancer patients. Specifically, therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.
A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar ...epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing
and
(FABP4
), and one highly expressing
and
(SPP1
). SPP1
macrophages in fibrotic lower lobes showed highly upregulated
and
expression. Low-level local proliferation of SPP1
macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1
macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1
macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease.
Systemic sclerosis (SSc) is a multi-organ autoimmune disease characterized by skin fibrosis. Internal organ involvement is heterogeneous. It is unknown whether disease mechanisms are common across ...all involved affected tissues or if each manifestation has a distinct underlying pathology.
We used consensus clustering to compare gene expression profiles of biopsies from four SSc-affected tissues (skin, lung, esophagus, and peripheral blood) from patients with SSc, and the related conditions pulmonary fibrosis (PF) and pulmonary arterial hypertension, and derived a consensus disease-associate signature across all tissues. We used this signature to query tissue-specific functional genomic networks. We performed novel network analyses to contrast the skin and lung microenvironments and to assess the functional role of the inflammatory and fibrotic genes in each organ. Lastly, we tested the expression of macrophage activation state-associated gene sets for enrichment in skin and lung using a Wilcoxon rank sum test.
We identified a common pathogenic gene expression signature-an immune-fibrotic axis-indicative of pro-fibrotic macrophages (MØs) in multiple tissues (skin, lung, esophagus, and peripheral blood mononuclear cells) affected by SSc. While the co-expression of these genes is common to all tissues, the functional consequences of this upregulation differ by organ. We used this disease-associated signature to query tissue-specific functional genomic networks to identify common and tissue-specific pathologies of SSc and related conditions. In contrast to skin, in the lung-specific functional network we identify a distinct lung-resident MØ signature associated with lipid stimulation and alternative activation. In keeping with our network results, we find distinct MØ alternative activation transcriptional programs in SSc-associated PF lung and in the skin of patients with an "inflammatory" SSc gene expression signature.
Our results suggest that the innate immune system is central to SSc disease processes but that subtle distinctions exist between tissues. Our approach provides a framework for examining molecular signatures of disease in fibrosis and autoimmune diseases and for leveraging publicly available data to understand common and tissue-specific disease processes in complex human diseases.
Interstitial lung diseases (ILDs) associated with autoimmune diseases show characteristic signs of imaging. Radiologic signs are also used in the identification of ILDs with features suggestive of ...autoimmune disease that do not meet the criteria for a specific autoimmune disease. Radiologists play a key role in identifying these signs and assessing their relevance as part of multidisciplinary team discussions. A radiologist may be the first health care professional to pick up signs of autoimmune disease in a patient referred for assessment of ILD or with suspicion for ILD. Multidisciplinary team discussion of imaging findings observed during follow-up may inform a change in diagnosis or identify progression, with implications for a patient’s treatment regimen. This article describes the imaging features of autoimmune disease-related ILDs and the role of radiologists in assessing their relevance.
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